Methods of treatment for solid tumors

ABSTRACT

The invention provides methods that relate to a novel therapeutic strategy for the treatment of hematological malignancies and inflammatory diseases. In particular, the method comprises administration of a compound of formula I, 
     
       
         
         
             
             
         
       
         
         
           
             or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising such compound admixed with at least one pharmaceutically acceptable excipient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/763,991, filed Apr. 20, 2010, which claims priority from U.S.Provisional Application No. 61/171,047, filed Apr. 20, 2009, thecontents of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The invention is in the field of therapeutics and medicinal chemistry.In particular, the invention concerns methods of treatment for certainsolid tumors that include administration of certain quinazolinonederivatives.

BACKGROUND ART

Cell signaling via 3′-phosphorylated phosphoinositides has beenimplicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity. Theenzyme responsible for generating these phosphorylated signalingproducts, phosphatidylinositol 3-kinase (PI 3-kinase; PI3K), wasoriginally identified as an activity associated with viral oncoproteinsand growth factor receptor tyrosine kinases that phosphorylatesphosphatidylinositol (PI) and its phosphorylated derivatives at the3′-hydroxyl of the inositol ring.

PI 3-kinase activation, is believed to be involved in a range ofcellular responses including cell growth, differentiation, andapoptosis. FIG. 1 shows some cellular pathways by which PI3K(represented by p110 and p85) participates in solid tumor activation.

The initial purification and molecular cloning of PI3-kinase revealedthat it was a heterodimer consisting of p85 and p110 subunits. Fourdistinct Class I PI3Ks have been identified, designated PI3K α, β, δ,and γ, each consisting of a distinct p110 catalytic subunit and aregulatory subunit. More specifically, three of the catalytic subunits,i.e., p110α, p110β and p110δ, each interact with the same regulatorysubunit, p85; whereas p110γ interacts with a distinct regulatorysubunit, p101. The patterns of expression of each of these PI3Ks inhuman cells and tissues are also distinct.

Identification of the p110δ isoform of PI 3-kinase is described inChantry et al., J. Biol. Chem., 272:19236-41 (1997). It was observedthat the human p110δ isoform is expressed in a tissue-restrictedfashion. It is expressed at high levels in lymphocytes and lymphoidtissues, suggesting that the protein might play a role inPI3-kinase-mediated signaling in the immune system. The p110β isoform ofPI3K may also play a role in PI3K-mediated signaling in certain cancers.FIG. 2 illustrates the relative amounts of these isoforms of p110 in anumber of different cancer cell lines. Some solid tumors exhibit littleor no p110α, and many have low levels of p110δ, but all of the onestested showed significant levels of p110β.

There is a need for a treatment of PI3K-mediated disorders relating tocancers, inflammatory diseases, and autoimmune diseases. Quinazolinonecompounds have been described as generally useful for treating mainlyhematologic cancers that express relatively high levels of p110δ,because the quinazolinones are more active as inhibitors of p110δ. OtherPI3K inhibitors are under development for treatment of solid tumors, butthey appear to be non-selective inhibitors of several isoforms of p110,or inhibitors mainly of p110α. For example, XL-147 inhibits p110α andp110δ and p110γ with similar IC-50's according to Exelixis, and has 10×lower activity on p110β; BEZ235 is described as a pan-PI3K inhibitorthat also acts on mTOR; and GDC-0941 is described as a p110α inhibitorInhibitors with lower selectivity, or with higher levels of p110αactivity, could be expected to have off-target activities; p110α, forexample, is involved in regulation of glucose and insulin levels. Thepresent invention provides a specific isomer of one quinazolinonecompound that is particularly useful for the treatment of solid tumors.While it is more active on p110δ than other isoforms of PI3K, thiscompound's ability to treat solid tumors is believed to be due to itsrelatively high activity as an inhibitor of p110β combined with a highlevel of oral bioavailability, and it exhibits relatively low levels offunctional activity against p110α.

SUMMARY

The invention provides novel methods to treat certain solid tumors,using a compound of formula (I). In one aspect, the invention provides amethod of treating cancer in a subject comprising administering to saidsubject an optically active compound of formula I:

or a pharmaceutically acceptable salt thereof. The optically activecompound is predominantly the S-isomer shown here, though it may containas a minor component some proportion of the R enantiomer. Preferably thecompound used in the methods of the invention consists primarily of theS-isomer as further discussed herein.

The methods of the invention include delivery of this compound byvarious routes of administration, but preferably the compound isadministered orally.

The subject can be any mammal; in preferred embodiments the subject is ahuman.

Without being bound by theory, the antitumor activity of this compoundis believed to arise from its inhibition of p110β more than frominhibition of p110δ or p110α. It exhibited activity in a variety ofcancer cell lines that expressed little p110δ, and some that did notexpress significant amounts of p110α; but all of the tested cell linesexpressed p110β.

Moreover, compound I exhibited comparatively low functional activity onp110α in a cell-transformation system designed to measure functionalactivity of these kinases, but is a potent inhibitor of both p110β andp110δ in that assay. See Example 1. This chick embryo fibroblast (CEF)transformation system has been reported as a useful way to assess thefunctional activity of the PI3K signaling pathway. Denley, et al.,“Oncogenic signaling of class I PI3K isoforms,” Oncogene, vol. 27(18),2561-74 (2008). Transformation of CEF cells in the assay depends uponfunctional kinase activity. Kang, et al., Proc. Nat'l Acad. Sci. USA,vol. 103(5), 1289-94 (2006). Similarly in other functional cell-basedassays, Compound I is most active on p110δ and p110β, with relativelylower activity against p110α.

As FIG. 4 illustrates, Compound I at 10 micromolar inhibitsphosphorylation of Akt, which is a downstream mediator of PI3Kactivation (See FIG. 1), in two cancer cell lines, T47D (breast cancer)and OVCAR-3 (ovarian cancer). It is significant, too, that T47D has amutation that activates p110α, yet that does not significantly reducethe effect of Compound I against this cell line, further suggesting thatthe antitumor activity of this compound must reside in its effect onother isoforms rather than on p110α. This distinguishes compound I fromother known PI3K inhibitors in development for treatment of solidtumors, which are believed to act primarily at the p110α isoform or onp110α plus other isoforms, or even on all PI3Ks plus mTOR.

Compound I is useful to treat certain cancers. In some embodiments thecancer is a non-hematopoietic cancer. In some embodiments, the cancer isa solid tumor selected from pancreatic cancer; bladder cancer;colorectal cancer; breast cancer; prostate cancer; renal cancer;hepatocellular cancer; lung cancer; ovarian cancer; cervical cancer;gastric cancer; esophageal cancer; head and neck cancer; melanoma;neuroendocrine cancers; CNS cancers; brain tumors; bone cancer; and softtissue sarcoma. In some embodiments it is lung cancer (non-small celllung cancer, small-cell lung cancer), colon cancer, CNS cancer,melanoma, ovarian cancer, renal cancer, prostate cancer or breastcancer.

In some embodiments, the method comprises administering an effectiveamount of compound I or a pharmaceutically acceptable salt of compoundI, to a subject afflicted with one of these cancers. In preferredembodiments, the compound is administered orally. The compound may beadministered alone or in the form of a pharmaceutical composition thatcomprises compound I admixed with at least one pharmaceuticallyacceptable excipient.

In particular embodiments, the cancer is breast cancer, lung cancer,prostate cancer, renal cancer, or ovarian cancer. In a particularembodiment, the method comprises administering to the subject to betreated, in addition to a compound of formula I, a therapeuticallyeffective amount of at least one additional therapeutic agent and/or anadditional therapeutic procedure selected to treat the cancer.

The invention thus provides a method of treating a solid tumor in asubject comprising administering to said subject an optically activecompound of formula I or a pharmaceutically acceptable salt thereof, ora pharmaceutical composition comprising an optically active compound ofFormula I or a pharmaceutically acceptable salt thereof, wherein theamount of the compound of Formula I or its salt is an amount effectiveto treat the solid tumor.

In certain embodiments, the solid tumor is selected from the groupconsisting of pancreatic cancer; bladder cancer; colorectal cancer;breast cancer; prostate cancer; renal cancer; hepatocellular cancer;lung cancer; ovarian cancer; cervical cancer; gastric cancer; esophagealcancer; head and neck cancer; melanoma; neuroendocrine cancers; CNScancers; brain tumors; bone cancer; and soft tissue sarcoma. In someembodiments, the solid tumor is selected from non-small cell lungcancer, small-cell lung cancer, colon cancer, CNS cancer, melanoma,ovarian cancer, renal cancer, prostate cancer and breast cancer.

For methods of the invention, compound I is optically active.Preferably, the S-enantiomer predominates over the R enantiomer by aratio of at least about 9:1. In specific embodiments, the S-enantiomerpredominates over the R enantiomer by a ratio of at least about 19:1.

In preferred embodiments, Compound I is administered orally. Typically,it is administered in a solid form, and commonly it is admixed with apharmaceutically acceptable diluent or excipient.

The method is applicable to the treatment of a variety of tumor types.In some embodiments, the cancer is ovarian, renal, breast, lung, colonor prostate cancer.

The subject is a mammal, and is typically a human. In some embodiments,the subject is refractory to chemotherapy treatment, or is in relapseafter treatment with chemotherapy. The methods of the invention are alsouseful to reduce the level of activity of p110β in the subject.

The compound of Formula I can be administered at a dose of 20-500mg/day. In some embodiments, the compound of formula I is administeredat least twice daily. In specific embodiments, it is administered at adose of 50-250 mg/day. In some embodiments, it is administered at a doseof 50-150 mg twice per day.

In some embodiments, the dose of Compound I is selected to provide aconcentration of compound I in the blood that reaches a point between 40and 10,000 ng/mL over a 12 hour period from the time of administration.In some embodiments, the dosing provides a concentration of compound Iin the blood that is between about 100 ng/mL and 6000 ng/mL in thetreated subject. In some embodiments, dosing is selected to produce aCmax (peak plasma level) of Compound I between 1000 ng/mL and 8,000ng/mL.

Compound I can be administered orally, transdermally, or by injection orinhalation. In some embodiments, it is administered orally.

In another aspect, the invention provides a combination therapy fortreating cancer, comprising administering Compound I to a subject who isconcurrently receiving treatment with an additional therapeutic agent,or an additional cancer therapy.

In some embodiments, the additional therapeutic agent to be used alongwith Compound I is selected from the following group consisting ofDocetaxel, Mitoxantrone, Prednisone, Estramustine, Anthracyclines,(doxorubicin (Adriamycin), epirubicin (Ellence), and liposomaldoxorubicin (Doxil)), Taxanes (docetaxel (Taxotere), paclitaxel (Taxol),and protein-bound paclitaxel (Abraxane)), Cyclophosphamide (Cytoxan),Capecitabine (Xeloda) and 5 fluorouracil (5 FU), Gemcitabine (Gemzar),methotrexate, Vinorelbine (Navelbine), an EGFR inhibitor such aserlotinib, Trastuzumab, Herceptin, Avastin, Platins (cisplatin,carboplatin), Temazolamide, Interferon alpha, and IL-2. In someembodiments, it is selected from the group consisting of an EGFRinhibitor, an mTOR inhibitor, a platin, and a taxane.

In some embodiments, the therapeutic procedure to be used along withCompound I is selected from the group consisting of peripheral bloodstem cell transplantation, autologous hematopoietic stem celltransplantation, autologous bone marrow transplantation, antibodytherapy, biological therapy, enzyme inhibitor therapy, total bodyirradiation, infusion of stem cells, bone marrow ablation with stem cellsupport, in vitro-treated peripheral blood stem cell transplantation,umbilical cord blood transplantation, immunoenzyme technique,immunohistochemistry staining method, pharmacological study, low-LETcobalt-60 gamma ray therapy, bleomycin, conventional surgery, radiationtherapy, high-dose chemotherapy and nonmyeloablative allogeneichematopoietic stem cell transplantation.

In some embodiments, the methods of the invention further compriseobtaining a biological sample from said subject; and analyzing thebiological sample with an analytical procedure selected from the groupconsisting of blood chemistry analysis, chromosomal translocationanalysis, needle biopsy, fluorescence in situ hybridization, laboratorybiomarker analysis, immunohistochemistry staining method, flow cytometryor a combination thereof. Analysis provides information that can be usedto determine whether to adjust the dose of Compound I up or down, or toterminate treatment with Compound I, or to add an additional therapeuticagent or therapeutic procedure to the treatment methods using CompoundI.

In some embodiments, Compound I is administered twice daily for about 28days, and is then discontinued for at least 7 days.

The following detailed description is to aid in understanding andemploying the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows part of the PI3K signaling pathway associated with solidtumor activation.

FIG. 2 shows the relative levels of the alpha, beta, delta and gammaisoforms of p110 in six different cancer cell lines, along with levelsof Akt and pAkt.

FIG. 3 shows the readout of a functional CEF transformation assay forthe relative activity of various isoforms of p110.

FIG. 4 a-b shows how Compound I, at concentrations from 0.01 uM to 10uM, affects phosphorylation of Akt, GSK13, and S6 in two cancer celllines, compared to how GDC-0941, which is described as a p110alphainhibitor affects the same phosphorylations.

FIG. 5 illustrates a reaction scheme for synthesis of Compound I.

FIG. 6 shows plasma levels of Compound I in mice that received a singleoral dose of the compound, compared to plasma levels of anotherquinazolinone compound (Compound B) with a similar structure, toillustrate the high oral bioavailability provided by Compound I.

FIG. 7 a-b shows dose-dependent inhibition of growth of tumor cellcultures for two different tumor lines.

FIG. 8 shows that Compound I at 30 mg/kg BID completely inhibited growthof a tumor xenograft over a five week period, while the correspondingxenografts in untreated control animals more than doubled in volume overthe same time period.

FIG. 9 shows that Compound I at 30 mg/kg BID significantly inhibitedgrowth of a tumor xenograft over a three week period, while thecorresponding xenografts in untreated control animals expanded much morerapidly during the same time period.

FIG. 10 shows the plasma concentration profile for Compound I in thexenograft-bearing mice of FIGS. 8 and 9, when administered as a singledose of 30 mg/kg.

FIG. 11 shows the plasma concentration profiles on the first and lastdays of multi-day testing of compound I in healthy mice receiving 60mg/kg, 120 mg/kg, or 240 mg/kg per day. The 60 mg/kg dose was welltolerated, demonstrating that the treatment dose (30 mg/kg BID) is bothtolerated and effective in mice.

MODES OF CARRYING OUT THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

The discussion of the general methods given herein is intended forillustrative purposes only. Other alternative methods and embodimentswill be apparent to those of skill in the art upon review of thisdisclosure.

A group of items linked with the conjunction “or” should not be read asrequiring mutual exclusivity among that group, but rather should also beread as “and/or” unless expressly stated otherwise. Although items,elements, or components of the invention may be described or claimed inthe singular, the plural is contemplated to be within the scope thereofunless limitation to the singular is explicitly stated.

The invention provides methods that relate to a novel therapeutic methodfor the treatment of cancer and particularly solid tumors. The inventioncomprises administering to said subject a compound of formula I:

or a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof, optionally admixed with at least onepharmaceutically acceptable excipient.

Compound I for use in the methods described herein is optically active,meaning it consists of predominantly one of two enantiomers. Thecompound has a single chiral center, in the noncyclic linking groupbetween the quinazolinone moiety and the purine moiety. The chiralcenter of the preferred enantiomer of the compound of Formula (I) is theS-isomer depicted above. The compound is used in optically active form,which contains predominantly the S-enantiomer. This compound may besynthesized in optically active form, or it may be prepared in racemicform (containing equal amounts of R and S isomers), and then the isomersmay be separated. A chiral synthesis of Compound I that provides the Senantiomer in very high optical purity is depicted herein. See FIG. 5.While it is preferable to substantially exclude the enantiomeric Risomer from the compound of Formula (I) for purposes of the invention,the methods can be practiced with mixtures of R and S isomers, providedthe S isomer is the major component of the mixture. Typically suchmixture will contain no more than about 10% of the R isomer, meaning theratio of S to R isomers is at least about 9:1, and preferably less than5% of the R-isomer, meaning the ratio of S to R enantiomers is at leastabout 19:1. In some embodiments the compound used has less than 2% Renantiomer, meaning it has an enantiomeric excess of at least about 96%.

The methods of the invention utilize an optically active form ofCompound I (the compound of Formula I), meaning in each instance, thecompound is optically active and contains predominantly theS-enantiomer, although it may contain the R-enantiomer of Compound I asa minor component. For clarity, where a dosage of a compound of FormulaI, or a dosage of Compound I is described herein, the dosage refers tothe weight of the compound of Formula I, including each enantiomer thatis present. Thus a dosage of 100 mg of Compound I as used herein, forexample, refers to the weight of the mixture of enantiomers rather thanthe weight of the S-enantiomer specifically. It could, for example,refer to 100 mg of a 9:1 mixture of S and R enantiomers, which wouldcontain about 90 mg of the S enantiomer, or to 100 mg of a 19:1 mixtureof S and R enantiomers, which would contain about 95 mg of the Senantiomer.

The methods of the invention are useful to treat cancers, particularlysolid tumors. In some embodiments, the cancer is a solid tumor selectedfrom pancreatic cancer; bladder cancer; colorectal cancer; breastcancer; prostate cancer; renal cancer; hepatocellular cancer; lungcancer; ovarian cancer; cervical cancer; gastric cancer; esophagealcancer; head and neck cancer; melanoma; neuroendocrine cancers; CNScancers; brain tumors; bone cancer; and soft tissue sarcoma. In someembodiments it is lung cancer (non-small cell lung cancer, small-celllung cancer), colon cancer, CNS cancer, melanoma, ovarian cancer, renalcancer, prostate cancer or breast cancer.

The efficacy of compound I is believed to arise from its in vivoinhibition of p110β activity primarily, though it also inhibits p110δactivity. Compound I is selective for inhibition of p110β and p110δ overp110α, and is selective for these two kinases over other kinases againstwhich it has been tested. Its selectivity is illustrated by its activityin a cellular assay of functional activity, where it inhibited p110βwith an EC-50 of about 150 nM, and p110δ with an EC-50 of about 15 nM,while showing much less activity on p110α (EC-50 was above 2000 nM).Even though its activity against p110β is lower than its activity onp110δ, because p110β is the dominant isoform of p110 that is observed insolid tumors, it is believed that the activity on the delta isoform isless important to its solid tumor activity than its activity on p110β.This is also consistent with the observation that Compound I exhibitsactivity against tumors that express little or no p110δ (suggesting theydo not rely on it), and against tumor cell lines where p110α isactivated (see T47D discussion above), suggesting that high levels ofthe alpha isoform do not reduce sensitivity to Compound I.

Selectivity with respect to p110α is important to the safety profile ofCompound I: p110α plays an essential role in insulin signaling andglucose metabolism. Nonselective PI3K inhibitors that also inhibit p110αactivity are expected to cause side effects or off-target adverseeffects by affecting insulin signaling and/or glucose metabolism, whichdo not seem to occur with Compound I. This is believed to contribute toreduction of off-target effects for Compound I.

Compound I is also selective for these PI3K isoforms over other lipidkinases, including other PI3K kinases, DNA-PK (another serine-threoninekinase), and mTOR. This table provides IC-50's for inhibition of kinaseactivity of these other lipid kinases:

PIKC3 2500 nM DNA-PK 13,000 nM mTOR 100,000 nM

Moreover, compound I has comparatively low activity on p110α in acell-transformation system designed to measure functional activity ofthese kinases, but is a potent inhibitor of both p110β and p110δ in thatassay. See Example 1. This chick embryo fibroblast (CEF) transformationsystem has been reported as a useful way to assess the functionalactivity of the PI3K signaling pathway. Denley, et al., “Oncogenicsignaling of class I PI3K isoforms,” Oncogene, vol. 27(18), 2561-74(2008). Transformation of CEF cells in the assay depends upon functionalkinase activity. Kang, et al., Proc. Nat'l Acad. Sci. USA, vol. 103(5),1289-94 (2006). The readout of this assay is based on the frequency oftransformation of CEF cells exposed to viral vectors carrying a specificp110 isoform of interest. See FIG. 3.

In this system, an EC-50 for functional activity of p110α was notreached at the highest concentration of Compound I tested (2000 nM); theEC-50 for inhibition of functional activity of p110β by Compound I wasabout 150 nM; and the EC-50 for inhibition of functional activity ofp110δ by Compound I was about 15 nM.

Similarly, other functional assays of inhibition of specific isoforms incell-based tests showed compound Ito have higher activity on the deltaand beta isoforms of p110 than on p110α. The p110α assay used SW3T3cells stimulated by PDGF, and p110 kinase activity was measured by Aktphosphorylation. The p110β activity was measured by lysophosphatidicacid stimulation of Akt phosphorylation in mouse embryonic fibroblasts.The activity of p110δ was measured by anti-FcεR1 antibody cross linkingstimulation of CD63 movement to the surface of basophils. Finally, theactivity of p110γ was measured by fMLP stimulation of CD63 antigenmovement to the cell surface of basophils. Again, compound I showedlittle inhibition of the alpha isoform, and was most active on the deltaand beta isoforms.

Cell-based assay p110α >20,000 EC₅₀ (nM) p110β    1,200 p110δ FB/WB8.4/19  p110γ 3,000/5,400

As FIG. 4 illustrates, Compound I at 10 micromolar inhibitsphosphorylation of Akt, which is a downstream mediator of PI3Kactivation (See FIG. 1), in two cancer cell lines, T47D (breast cancer)and OVCAR-3 (ovarian cancer). It is significant to note that T47D has amutation that activates p110α, yet compound I provides good activityagainst this cell line, further demonstrating that the antitumoractivity of this compound most likely resides in its effect on otherisoforms rather than on p110α.

Bioavailability of Compound I upon oral administration is especiallygood, even compared to other quinazolinones of similar structures. Forexample, FIG. 6 illustrates that Compound I produces higher plasmaconcentrations of drug than another quinazolinone compound having asimilar structure (Compound B). Note that Compound I was administeredorally to mice at half the dosage of Compounds B, but produced a higherpeak plasma level. The degree of difference in oral bioavailabilitybetween Compound I and Compound B observed in this test is surprising.

Treatments of the invention typically involve administration of compoundIto a subject in need of treatment on a daily basis for at least oneweek or more than one week, often for 2-4 weeks, and sometimes for 1-3months or more. The half-life of Compound I in vivo in mice and rats isseveral hours—see FIG. 6. It is thus sometimes desirable to administercompound I in multiple doses each day, in order to maintain efficaciousplasma levels over a prolonged period of time. Administration may bedone in two doses per day, or three doses per day, or in someembodiments, four doses per day or more, particularly when Compound I isadministered orally. Alternatively, Compound I can be administeredintravenously at a rate that maintains an efficacious plasma level for aprolonged period of time. Suitably, it would be administered at a rateto achieve a plasma level of at least about 1 micromolar, or at least 3micromolar, or at least 5 micromolar. FIG. 6 shows that plasma levels ofabout 500 ng/mL (over 1 micromolar) can be maintained for several hoursfollowing a single oral dose of 20 mg/kg of Compound I; thisdemonstrates that high plasma levels of Compound I, e.g., concentrationsconsistent with the levels shown to be effective in functional assays,can be achieved with tolerated doses of compound I.

Compound I has been shown to induce apoptosis of a variety of solidtumor cells. FIG. 7 shows its dose-dependent inhibition of tumor cellculture growth, measured by optical density at 459 nm, for a breastcancer cell line (T47D) and an ovarian cancer cell line (OVCAR-3). Itdemonstrates that exposure to 5-10 micromolar levels of Compound Iprovides strong inhibition of growth in cell cultures.

FIG. 8 shows a dose-dependent inhibition of growth of an ovarian cancerxenograft tumor, as judged by measuring tumor volume, upon treatmentwith Compound I. Tumor volume actually decreased during a treatmentlasting over 30 days in treated animals receiving 30 mg/kg Compound I,BID, while tumor volume more than doubled in the untreated controlanimals during the same time period. This demonstrates that Compound Iis effective to treat a solid tumor in vivo.

Similarly, FIG. 9 shows efficacy of Compound I for treating anothersolid tumor xenograft (A498, a human renal cancer cell line). As theFigure shows, treatment of mice bearing A498 tumors with Compound I at30 mg/kg BID for 20 days produced effective antitumor activity in vivo.While tumor volume approximately doubled over this time in the treatedanimals, it increased more than 5-fold in the untreated animals. Again,this shows compound I is effective to treat a solid tumor in vivo.

FIG. 10 shows plasma concentrations of Compound I in the mice bearingeach of the tumor xenografts used for FIGS. 8 and 9, following a singleoral dose of Compound I at 30 mg/kg. At this range, which was theeffective dosage used in the tests shown in FIGS. 8 and 9, plasmaconcentration of compound I reaches about 5000-7000 ng/mL.

FIG. 11 shows the plasma concentration profiles on the first and lastdays of multi-day testing of compound I in healthy mice receiving 60mg/kg, 120 mg/kg, or 240 mg/kg per day. The 60 mg/kg dose was welltolerated, demonstrating that the treatment dose (30 mg/kg BID) is bothtolerated and effective in mice.

Compound I has also been tested in a battery of tumor cell assays knownas the NCI panel. It demonstrated substantial growth inhibition of mostof the cancer cell lines in the panel, and was generally more active onthese cancer cell lines than Compound B, which was included forcomparison. The following Table shows the GI-50 (concentration providing50% growth inhibition, in μM) for each compound in these cell cultureassays.

Tumor Compound B Compound I Type Cell Line GI-50 (μM) GI-50 (μM) Non-A549 50.4 20.7 Small Cell EKVX 28.2 16.7 Lung HOP-62 39.7 14.8 CancerHOP-92 34.8 0.3 NCI-H226 100 100 NCI-H23 100 100 NCI-H322M 32.2 10.5NCI-H460 34.7 25.7 NCI-H522 52.7 51.8 Colon COLO205 30.1 33.8 CancerHCC-2998 53.3 32.4 HCT-116 54.3 37.9 HCT-15 48.4 30.3 HT29 39.1 16.5KM12 54.1 2 SW-620 86.4 69.3 CNS SF-268 29.8 1.89 Cancer SF-295 8.181.81 SF-539 28.6 15.9 SNB-19 54.8 19.3 SNB-75 2.96 0.0594 U251 70.4 59.6Melanoma LOX IMVI 31.1 36.1 MALME-3M 31.6 4.58 M14 58.4 88.9 SK-MEL-2100 100 SK-MEL-28 38.6 12.8 SK-MEL-5 32.9 31.3 UACC-257 33.5 17.4UACC-62 4.2 1.43 Ovarian IGROV1 3.54 2.7 OVCAR-3 15.5 0.316 OVCAR-4 100100 OVCAR-5 40.1 27.7 OVCAR-8 96 44.6 SK-OV-3 13.8 4.02 Renal 786-0 5.481.99 A498 2.38 0.615 ACHN 24.4 10.7 CAKI-1 13.4 1.01 RXF 393 74.2 1.14SN12C 11 22.5 TK-10 31.7 1.24 UO-31 5.32 2.01 Prostate PC-3 12 0.647DU-145 43.8 1.35 Breast MCF7 7.08 2.54 Cancer ADR-RES 77.9 58.6MDA-MB-231 100 100 HS 578T 6.91 2.24 MDA-MB-435 16.4 43.1 BT-549 14.30.538 T-47D 3.18 0.571 MDA-MB-468 13.6

As a means of comparing the overall activity of these two compounds ondiverse solid tumor cell types, the numbers of cell lines that have GI50values of 2 micromolar or less were determined for each compound; thesecell lines were considered particularly sensitive ones. Using thismeasure, 1.8% of cell lines were particularly sensitive to Compound B atthis level, while 39% were particularly sensitive to Compound I at thesame concentration. Despite structural similarity to Compound B, it isapparent that Compound I is much more active on solid tumors thancompound B.

Below is a table of the number of cell lines for each type of tumor thatwere found to have GI50 values less than 2 micromolar for Compound I.

Tumor Type Cell line number % EC₅₀ < 2 μm NSCLC 1/9 11% Colon 1/7 14%CNS 3/6 50% Melanoma 1/8 12% Ovarian 2/6 33% Prostate 2/2 100%  Renal6/8 75% Breast 5/8 62%

In a particular embodiment, the cancer is a solid tumor such as lungcancer (non-small cell lung cancer, small-cell lung cancer), coloncancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostatecancer or breast cancer. As the above table indicates, breast, renal,prostate and CNS cancers are particularly sensitive to compound I, so insome embodiments, the method is used to treat a subject having any oneof these cancers.

In a particular embodiment, the cancer is not a hematological cancer,e.g., it is not a lymphoma or leukemia or multiple myeloma. Exemplarysolid tumors treatable by the methods disclosed herein include breast,lung, colon, ovarian, renal, and prostate cancer.

In a particular embodiment, a compound of formula I is administered in atherapeutically effective amount, to a subject diagnosed with at leastone cancer disclosed as treatable by the methods herein.

The therapeutically effective amount can be determined by one ofordinary skill based on the subject's health, age, body weight, andcondition. In some embodiments, the amount is normalized to thesubject's body weight. For example, a dosage may be expressed as anumber of milligrams of Compound I per kilogram of the subject's bodyweight (mg/kg). Dosages of between about 0.1 and 100 mg/kg are oftenappropriate, and in some embodiments a dosage of between 0.5 and 60mg/kg is used. Normalizing according to the subject's body weight isparticularly useful when adjusting dosages between subjects of widelydisparate size, such as when converting an effective dosage in a dog toa dosage suitable for a human subject.

In other embodiments, the daily dosage may be described as a totalamount of Compound I administered per dose or per day. Daily dosage ofCompound I is typically between about 10 mg and 1000 mg. Whenadministered orally, the total daily dosage for a human subject istypically between about 50 mg and 750 mg.

In a particular embodiment, a compound of formula I is administered at adose of 20-500 mg/day.

In a particular embodiment, a compound of formula I is administered at adose of 50-250 mg/day.

In a particular embodiment, a compound of formula I is administered at adose of 25 to 150 mg per dose, and two to four doses are administeredper day (e.g., BID dosing with 25 to 150 mg doses, or TID dosing withdoses between 25 and 150 mg, or QID dosing with doses between 25 and 150mg). In a preferred embodiment, a subject is treated with 50 mg to 100mg doses of Compound I twice per day, or 50-100 mg doses three times perday, or 50-100 mg doses four times per day.

Treatment with the compounds of the invention are frequently continuedfor a number of days; for example, commonly treatment would continue forat least 7 days, about 14 days, or about 28 days, for one cycle oftreatment. Treatment cycles are well known in cancer chemotherapy, andare frequently alternated with resting periods of 1-28 days, commonly 7days or 14 days, between cycles.

In a particular embodiment, the method comprises administering to saidsubject an initial daily dose of 20-500 mg of a compound of formula Iand increasing said dose by increments until clinical efficacy isachieved. Increments of about 25, 50, or 100 mg can be used to increasethe dose. The dosage can be increased daily, every other day, twice perweek, or once per week.

In a particular embodiment, this method comprises continuing to treatsaid subject by administering the compound of formula I at a dosagewhere clinical efficacy is achieved for a week or more, or reducing saiddose by increments to a level at which efficacy can be maintained.Efficacy can be monitored by conventional methods such as assessingtumor size or spreading (metastasis).

In a particular embodiment, the method comprises administering to saidsubject an initial daily dose of 20-500 mg of a compound of formula Iand increasing said dose to a total dosage of 50-400 mg per day over atleast 6 days. Optionally, the dosage can be further increased to about750 mg/day.

In a particular embodiment, a compound of formula I is administered atleast twice daily. In some embodiments the compound is administeredthree times per day. In some embodiments the compound is administeredfour times per day, or more than four times per day.

In a particular embodiment, the method comprises reducing the level ofPI3Kβ activity in the subject.

In a particular embodiment, the subject is a human subject. Typicallythe subject is a human diagnosed as having a cancer disclosed herein astreatable by compound I.

In a particular embodiment, the compound is administered at a rateselected to produce a concentration of compound in the blood betweenabout 40 ng/mL and 3,000 ng/mL, and maintaining such concentrationduring a 4-12 hour period following administration. In anotherparticular embodiment, the dose size and frequency are selected toachieve a concentration of compound in the blood that is between75-2,000 ng/mL and maintain that concentration during a 4-12 hour periodfrom the time of administration. In some embodiments, the dose size andfrequency are selected to achieve a concentration of compound in theblood that is between 100-1,000 ng/mL following administration. In someembodiments, the dose size and frequency are selected to achieve aconcentration of compound in the blood that is between 100-500 ng/mLover a 12 hour period from the time of administration. Desirably, thedose size and frequency are selected to achieve a C_(max), plasma levelof Compound I that is at least about 500 ng/mL and does not exceed about10,000 ng/mL.

In certain embodiments, Compound I is administered orally,intravenously, transdermally, or by inhalation. Preferably, the compoundis administered orally. In some embodiments, it is administered orallyin a dose of about 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 75 mg, or 100 mg,125 mg, 150 mg, or 200 mg per dose, and the dose may be administered ata frequency of once per day, twice per day, three times per day, or fourtimes per day.

In a particular embodiment, the method comprises administering inaddition to a compound of formula Ito said subject a therapeuticallyeffective amount of at least one additional therapeutic agent and/or atherapeutic procedure selected to treat said cancer or autoimmunedisease in said subject.

In a particular embodiment, said therapeutic agent is selected from thefollowing group consisting of Docetaxel, Mitoxantrone, Prednisone,Estramustine, Anthracyclines, (doxorubicin (Adriamycin), epirubicin(Ellence), and liposomal doxorubicin (Doxil)), Taxanes (docetaxel(Taxotere), paclitaxel (Taxol), and protein-bound paclitaxel(Abraxane)), Cyclophosphamide (Cytoxan), Capecitabine (Xeloda) and 5fluorouracil (5 FU), Gemcitabine (Gemzar), methotrexate, Vinorelbine(Navelbine), an EGFR inhibitor such as erlotinib, Trastuzumab(Herceptin, this drug is only of use in women whose breast cancers havethe HER-2 gene), Avastin, Platins (cisplatin, carboplatin),Temazolamide, Interferon alpha, and IL-2.

In a particular embodiment, said therapeutic agent is selected from thegroup consisting of an EGFR inhibitor, an mTOR inhibitor, and a taxane.

In a particular embodiment, the therapeutic procedure is selected fromthe group consisting of peripheral blood stem cell transplantation,autologous hematopoietic stem cell transplantation, autologous bonemarrow transplantation, antibody therapy, biological therapy, enzymeinhibitor therapy, total body irradiation, infusion of stem cells, bonemarrow ablation with stem cell support, in vitro-treated peripheralblood stem cell transplantation, umbilical cord blood transplantation,immunoenzyme technique, immunohistochemistry staining method,pharmacological study, low-LET cobalt-60 gamma ray therapy, bleomycin,conventional surgery, radiation therapy, high-dose chemotherapy andnonmyeloablative allogeneic hematopoietic stem cell transplantation.

In a particular embodiment, the method further comprises obtaining abiological sample from said subject; and analyzing said biologicalsample with an analytical procedure selected from the group consistingof blood chemistry analysis, chromosomal translocation analysis, needlebiopsy, fluorescence in situ hybridization, laboratory biomarkeranalysis, immunohistochemistry staining method, flow cytometry or acombination thereof. Analysis provides information about progression ofthe tumor or of the treatment, and is useful for determining dosages toadminister, for adjusting dosages during a treatment cycle, and fordeciding whether to continue or discontinue the treatments of theinvention.

In certain embodiments, the optically active compound used in themethods described herein is enriched with the S-enantiomer shown here,and preferably it is at least 90% S-enantiomer, containing no more thanabout 10% of the enantiomeric R isomer:

In some embodiments, the compound of Formula I used in the methodsdescribed herein is at least 80% the S-enantiomer, containing less than20% of its enantiomeric R-isomer In some embodiments the compound has anenantiomeric excess (e.e.) of at least 90% or at least 95% favoring theS-isomer.

In certain embodiments, the compound is primarily composed of theS-enantiomer, wherein this isomer comprises at least 66-95%, or about85-99% of the S-isomer, in excess over any R-enantiomer present. Incertain embodiments, the compound comprises at least 95% of theS-enantiomer. In the cellular and patient experiments provided in theExample section, the sample of compound I used was over 99% the Senantiomer, with less than 1% of the R enantiomer.

The term “selective PI3Kδ inhibitor” or “selective PI3Kβ inhibitor”,etc., as used herein, refers to a compound that inhibits the PI3Kδ orPI3Kβ isozyme, respectively, more effectively than at least one otherisozyme of the PI3K family. The selective inhibitor may also inhibitother isozymes of PI3K, but requires higher concentrations to achievethe same degree of inhibition of the other isozymes. “Selective” canalso be used to describe a compound that inhibits a particularPI3-kinase more so than a comparable compound. A “selective PI3Kδinhibitor” compound is understood to be more selective for PI3Kδ thancompounds conventionally and generically designated PI3K inhibitors,e.g., wortmannin or LY294002, which are considered non-selective PI3Kinhibitors.

“Treating” as used herein refers to inhibiting a disorder, i.e.,arresting its development; relieving the disorder, i.e., causing itsregression; or ameliorating the disorder, i.e., reducing the severity ofat least one of the symptoms associated with the disorder. In someembodiments, “treating” refers to preventing a disorder from occurringin an animal that can be predisposed to the disorder, but has not yetbeen diagnosed as having it. “Disorder” is intended to encompass medicaldisorders, diseases, conditions, syndromes, and the like, withoutlimitation.

In certain embodiments, the invention provides methods to treat a solidtumor, typically a non-hematopoietic carcinoma. In some embodiments, thecancer is a solid tumor selected from pancreatic cancer; bladder cancer;colorectal cancer; breast cancer; prostate cancer; renal cancer;hepatocellular cancer; lung cancer; ovarian cancer; cervical cancer;gastric cancer; esophageal cancer; head and neck cancer; melanoma;neuroendocrine cancers; CNS cancers; brain tumors; bone cancer; and softtissue sarcoma. In some embodiments it is lung cancer (non-small celllung cancer, small-cell lung cancer), colon cancer, CNS cancer,melanoma, ovarian cancer, renal cancer, prostate cancer or breastcancer. In some embodiments, the cancer is breast, lung, colon, renal,ovarian, or prostate cancer.

In certain embodiments, the invention provides methods to treat a solidtumor that is associated with abnormal or undesirable cellular signalingactivity mediated by PI3Kβ. In certain embodiments, the solid tumor isselected from the group consisting of pancreatic cancer; bladder cancer;colorectal cancer; breast cancer, including metastatic breast cancer;prostate cancer, including androgen-dependent and androgen-independentprostate cancer; renal cancer, including, e.g., metastatic renal cellcarcinoma; hepatocellular cancer; lung cancer, including, e.g.,non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC),and adenocarcinoma of the lung; ovarian cancer, including, e.g.,progressive epithelial or primary peritoneal cancer; cervical cancer;gastric cancer; esophageal cancer; head and neck cancer, including,e.g., squamous cell carcinoma of the head and neck; melanoma;neuroendocrine cancer, including metastatic neuroendocrine tumors; braintumors, including, e.g., glioma, anaplastic oligodendroglioma, adultglioblastoma multiforme, and adult anaplastic astrocytoma; bone cancer;and soft tissue sarcoma.

In one embodiment, the cancer to be treated with the methods describedherein is a solid tumor that exhibits a functional loss of PTEN(phosphatase and tensin homolog, a phosphatase that acts as a tumorsuppressor) activity. Loss of PTEN activity often occurs in cancers, andenhances the sensitivity of a tumor to PI3K inhibitors. The NCI panelcontains a number of cell lines known to have mutations in PTEN, and 70%of those cell lines were inhibited by Compound I, and two of the onesthat were not sensitive to Compound I proved to have no functional lossof PTEN activity. The following Table summarizes the cell lines found tobe sensitive to Compound I and the known mutations in those cell lines.Of these mutations, only PTEN was found to be significantly correlatedwith efficacy of Compound I (p<0.036).

Tumor Type CDKN2 TP53 PTEN PI3KCA BRAF HRAS SMAD4 BRCAI Hop-92 X X KM12X X X SF-268 X X SF-295 X X SNB-75 X UACC-62 X X X IGROV-1 X X X XOVCAR-3 X 786-0 X X X A498 X CAK1-1 X RXF-393 X X X TK-10 X UO-31 X PC-3X X DU-145 _((RB1)) X X MCF7 X E545K HS-578T X X BT-549 _((RB1)) X T47DX H1047R 50% 75% 35% 10% 5% 5% 5% 10%

Accordingly, solid tumors with significantly reduced PTEN phosphataseactivity are particularly suitable for treatment with compound I. TheWellcome Trust Sanger Institute recently published information on theincidence of PTEN mutations in primary tumor tissues, indicating thatbreast, CNS, cervix, endometrial, kidney, ovary, prostate, skin, testis,and urinary tract tumors frequently include PTEN mutations. Accordingly,in some embodiments, the methods of the invention are used to treat asubject afflicted with one or more of these particular cancers, or aPTEN-deficient cancer selected from breast, CNS, cervix, endometrial,kidney, ovary, prostate, skin, testis, and urinary tract tumors.

In certain embodiments, the method described herein is useful intargeting cells mediating Akt phosphorylation, because compound Iinhibits Akt phosphorylation as illustrated in FIG. 4.

For the treatment of a solid tumor, it is advantageous that the compoundof Formula I exhibits good activity against p110β, since solid tumorsoften utilize this isozyme rather than or more than p110δ. Thus in someembodiments, the solid tumor is one that expresses p110β at a higherlevel than its level of expression of p110δ. In some embodiments, thesolid tumor is one with a low level of p110δ activity, such as oneexpressing less than about 20% as much p110δ as p110β.

In some embodiments, the subject for treatments described herein is onewho has been diagnosed with at least one of the cancers described hereinas treatable by the use of a compound of Formula I. In some embodiments,the subject has been diagnosed with a cancer named herein, and hasproven refractory to treatment with at least one conventionalchemotherapeutic agent. Thus in one embodiment, the treatments of theinvention are directed to patients who have received one or more thanone such treatment and remain in need of more effective treatment.

In one embodiment, the method described herein comprises administeringto a subject a compound of formula I described herein, in combinationwith a therapy used to treat cancer. The “therapy” used to treat cancer,as used herein, is any well-known or experimental form of treatment usedto treat cancer that does not include the use of a compound of formulaI. In certain embodiments, the combination of a compound of formula Iwith a conventional or experimental therapy used to treat cancerprovides beneficial and/or desirable treatment results superior toresults obtained by treatment without the combination. In certainembodiments, said therapies used to treat cancer are well-known to aperson having ordinary skill in the art and are described in theliterature. Therapies include, but are not limited to, chemotherapy,combinations of chemotherapy, biological therapies, immunotherapy,radioimmunotherapy, and the use of monoclonal antibodies, and vaccines.In certain embodiments, the combination method provides for a compoundof formula I administered simultaneously or during the period ofadministration of the therapy. In certain embodiments, the combinationmethod provides for a compound of formula I administered prior to orafter the administration of the therapy. The exact details regarding theadministration of the combination may be determined experimentally. Therefinement of sequence and timing of administering a compound of formulaI with a selected therapy will be tailored to the individual subject,the nature of the condition to be treated in the subject, and generally,the judgment of the attending practitioner.

Additional therapeutic agents for combinations with Compound I includethose routinely used in the treatment of solid tumors, particularlyDocetaxel, Mitoxantrone, Prednisone, Estramustine, Anthracyclines,(doxorubicin (Adriamycin), epirubicin (Ellence), and liposomaldoxorubicin (Doxil)), Taxanes (docetaxel (Taxotere), paclitaxel (Taxol),and protein-bound paclitaxel (Abraxane)), Cyclophosphamide (Cytoxan),Capecitabine (Xeloda) and 5 fluorouracil (5 FU), Gemcitabine (Gemzar),methotrexate, Vinorelbine (Navelbine), an EGFR inhibitor such aserlotinib, Trastuzumab (Herceptin, this drug is only of use in womenwhose breast cancers have the HER-2 gene), Avastin, Platins (cisplatin,carboplatin), Temazolamide, Interferon alpha, and IL-2.

In certain embodiments, the method comprises administering to saidsubject, in addition to an effective amount of compound I, at least onetherapeutic agent and/or therapeutic procedure selected to treat saidcancer in said subject. In certain embodiments, the method comprisesadministering in addition to a compound of Ito said subject, atherapeutically effective amount of an additional therapeutic agentselected from Docetaxel, Mitoxantrone, Prednisone, Estramustine,Anthracyclines, (doxorubicin (Adriamycin), epirubicin (Ellence), andliposomal doxorubicin (Doxil)), Taxanes (docetaxel (Taxotere),paclitaxel (Taxol), and protein-bound paclitaxel (Abraxane)),Cyclophosphamide (Cytoxan), Capecitabine (Xeloda) and 5 fluorouracil (5FU), Gemcitabine (Gemzar), methotrexate, Vinorelbine (Navelbine), anEGFR inhibitor such as erlotinib, Trastuzumab (Herceptin, this drug isonly of use in women whose breast cancers have the HER-2 gene), Avastin,Platins (cisplatin, carboplatin), Temazolamide, Interferon alpha, andIL-2.

The compounds of the invention may be formulated for administration toanimal subject using commonly understood formulation techniques wellknown in the art. Formulations which are suitable for particular modesof administration and for the compounds of formula I may be found inRemington's Pharmaceutical Sciences, latest edition, Mack PublishingCompany, Easton, Pa.

A compound of the present invention can be administered as the neatchemical, but it is typically preferable to administer the compound inthe form of a pharmaceutical composition or formulation. Accordingly,the present invention also provides pharmaceutical compositions thatcomprise a compound of formula I and a biocompatible pharmaceuticalcarrier, adjuvant, or vehicle. The composition can include the agent asthe only active moiety or in combination with other agents, such asoligo- or polynucleotides, oligo- or polypeptides, drugs, or hormonesmixed with excipient(s) or other pharmaceutically acceptable carriers.Carriers and other ingredients can be deemed pharmaceutically acceptableinsofar as they are compatible with other ingredients of the formulationand not deleterious to the recipient thereof.

The pharmaceutical compositions are formulated to contain suitablepharmaceutically acceptable carriers, and can optionally compriseexcipients and auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically. Theadministration modality will generally determine the nature of thecarrier. For example, formulations for parenteral administration cancomprise aqueous solutions of the active compounds in water-solubleform. Carriers suitable for parenteral administration can be selectedfrom among saline, buffered saline, dextrose, water, and otherphysiologically compatible solutions. Preferred carriers for parenteraladministration are physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Fortissue or cellular administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art. For preparations comprisingproteins, the formulation can include stabilizing materials, such aspolyols (e.g., sucrose) and/or surfactants (e.g., nonionic surfactants),and the like.

Alternatively, formulations for parenteral use can comprise dispersionsor suspensions of the active compounds prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, and synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions can contain substances that increase the viscosity of thesuspension, such as sodium carboxy-methylcellulose, sorbitol, ordextran. Optionally, the suspension also can contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions. Aqueouspolymers that provide pH-sensitive solubilization and/or sustainedrelease of the active agent also can be used as coatings or matrixstructures, e.g., methacrylic polymers, such as the EUDRAGIT™ seriesavailable from Rohm America Inc. (Piscataway, N.J.). Emulsions, e.g.,oil-in-water and water-in-oil dispersions, also can be used, optionallystabilized by an emulsifying agent or dispersant (surface activematerials; surfactants). Suspensions can contain suspending agents suchas ethoxylated isostearyl alcohols, polyoxyethlyene sorbitol andsorbitan esters, microcrystalline cellulose, aluminum metahydroxide,bentonite, agar-agar, gum tragacanth, and mixtures thereof.

Liposomes containing the active agent also can be employed forparenteral administration. Liposomes generally are derived fromphospholipids or other lipid substances. The compositions in liposomeform also can contain other ingredients, such as stabilizers,preservatives, excipients, and the like. Preferred lipids includephospholipids and phosphatidyl cholines (lecithins), both natural andsynthetic. Methods of forming liposomes are known in the art. See, e.g.,Prescott (Ed.), METHODS IN CELL BIOLOGY, Vol. XIV, p. 33, AcademicPress, New York (1976).

The pharmaceutical compositions comprising the agent in dosages suitablefor oral administration can be formulated using pharmaceuticallyacceptable carriers well known in the art. The preparations formulatedfor oral administration can be in the form of tablets, pills, capsules,cachets, dragees, lozenges, liquids, gels, syrups, slurries, elixirs,suspensions, or powders. To illustrate, pharmaceutical preparations fororal use can be obtained by combining the active compounds with a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragée cores. Oral formulations can employ liquidcarriers similar in type to those described for parenteral use, e.g.,buffered aqueous solutions, suspensions, and the like.

Preferred oral formulations include tablets, dragees, and gelatincapsules. These preparations can contain one or excipients, whichinclude, without limitation:

a) diluents, such as sugars, including lactose, dextrose, sucrose,mannitol, or sorbitol;

b) binders, such as magnesium aluminum silicate, starch from corn,wheat, rice, potato, etc.;

c) cellulose materials, such as methylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose, polyvinylpyrrolidone,gums, such as gum arabic and gum tragacanth, and proteins, such asgelatin and collagen;

d) disintegrating or solubilizing agents such as cross-linked polyvinylpyrrolidone, starches, agar, alginic acid or a salt thereof, such assodium alginate, or effervescent compositions;

e) lubricants, such as silica, talc, stearic acid or its magnesium orcalcium salt, and polyethylene glycol;

f) flavorants and sweeteners;

g) colorants or pigments, e.g., to identify the product or tocharacterize the quantity (dosage) of active compound; and

h) other ingredients, such as preservatives, stabilizers, swellingagents, emulsifying agents, solution promoters, salts for regulatingosmotic pressure, and buffers.

In some preferred oral formulations, the pharmaceutical compositioncomprises at least one of the materials from group (a) above, or atleast one material from group (b) above, or at least one material fromgroup (c) above, or at least one material from group (d) above, or atleast one material from group (e) above. Preferably, the compositioncomprises at least one material from each of two groups selected fromgroups (a)-(e) above.

Gelatin capsules include push-fit capsules made of gelatin, as well assoft, sealed capsules made of gelatin and a coating such as glycerol orsorbitol. Push-fit capsules can contain the active ingredient(s) mixedwith fillers, binders, lubricants, and/or stabilizers, etc. In softcapsules, the active compounds can be dissolved or suspended in suitablefluids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycol with or without stabilizers.

Dragée cores can be provided with suitable coatings such as concentratedsugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.

The pharmaceutical composition can be provided as a salt of the activeagent. Salts tend to be more soluble in aqueous or other protonicsolvents than the corresponding free acid or base forms.Pharmaceutically acceptable salts are well known in the art. Compoundsthat contain acidic moieties can form pharmaceutically acceptable saltswith suitable cations. Suitable pharmaceutically acceptable cationsinclude, for example, alkali metal (e.g., sodium or potassium) andalkaline earth (e.g., calcium or magnesium) cations.

Compounds of structural formula (I) that contain basic moieties can formpharmaceutically acceptable acid addition salts with suitable acids. Forexample, Berge, et al., describe pharmaceutically acceptable salts indetail in J. Pharm. Sci., 66:1 (1977). The salts can be prepared in situduring the final isolation and purification of the compounds of theinvention or separately by reacting a free base function with a suitableacid.

Representative acid addition salts include, but are not limited to,acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorolsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate (isothionate), lactate, maleate,methanesulfonate or sulfate, nicotinate, 2-naphthalenesulfonate,oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate,pivalate, propionate, succinate, tartrate, thiocyanate, phosphate orhydrogen phosphate, glutamate, bicarbonate, p-toluenesulfonate, andundecanoate. Examples of acids that can be employed to formpharmaceutically acceptable acid addition salts include, withoutlimitation, such inorganic acids as hydrochloric acid, hydrobromic acid,sulfuric acid, and phosphoric acid, and such organic acids as oxalicacid, maleic acid, succinic acid, and citric acid.

Basic nitrogen-containing groups can be quaternized with such agents aslower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl,and diamyl sulfates; long chain alkyl halides such as decyl, lauryl,myristyl, and stearyl chlorides, bromides, and iodides; arylalkylhalides such as benzyl and phenethyl bromides; and others. Productshaving modified solubility or dispersibility are thereby obtained.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier can be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Accordingly, there also is contemplated an article ofmanufacture, such as a container comprising a dosage form of a compoundof the invention and a label containing instructions for use of thecompound. Kits are also contemplated under the invention. For example,the kit can comprise a dosage form of a pharmaceutical composition and apackage insert containing instructions for use of the composition intreatment of a medical condition. In either case, conditions indicatedon the label can include treatment of inflammatory disorders, cancer,etc.

Methods of Administration

Pharmaceutical compositions comprising a compound of formula I can beadministered to the subject by any conventional method, includingparenteral and enteral techniques. Parenteral administration modalitiesinclude those in which the composition is administered by a route otherthan through the gastrointestinal tract, for example, intravenous,intraarterial, intraperitoneal, intramedullarly, intramuscular,intraarticular, intrathecal, and intraventricular injections. Enteraladministration modalities include, for example, oral (including buccaland sublingual) and rectal administration. Transepithelialadministration modalities include, for example, transmucosaladministration and transdermal administration. Transmucosaladministration includes, for example, enteral administration as well asnasal, inhalation, and deep lung administration; vaginal administration;and rectal administration. Transdermal administration includes passiveor active transdermal or transcutaneous modalities, including, forexample, patches and iontophoresis devices, as well as topicalapplication of pastes, salves, or ointments. Parenteral administrationalso can be accomplished using a high-pressure technique, e.g.,POWDERJECT™.

Surgical techniques include implantation of depot (reservoir)compositions, osmotic pumps, and the like. A preferred route ofadministration for treatment of inflammation can be local or topicaldelivery for localized disorders such as arthritis, or systemic deliveryfor distributed disorders, e.g., intravenous delivery for reperfusioninjury or for systemic conditions such as septicemia. For otherdiseases, including those involving the respiratory tract, e.g., chronicobstructive pulmonary disease, asthma, and emphysema, administration canbe accomplished by inhalation or deep lung administration of sprays,aerosols, powders, and the like.

The compound of formula I can be administered before, during, or afteradministration of chemotherapy, radiotherapy, and/or surgery. Theformulation and route of administration chosen will be tailored to theindividual subject, the nature of the condition to be treated in thesubject, and generally, the judgment of the attending practitioner.

The therapeutic index of the compound of formula I can be enhanced bymodifying or derivatizing the compounds for targeted delivery to cancercells expressing a marker that identifies the cells as such. Forexample, the compounds can be linked to an antibody that recognizes amarker that is selective or specific for cancer cells, so that thecompounds are brought into the vicinity of the cells to exert theireffects locally, as previously described (see for example, Pietersz, etal., Immunol. Rev., 129:57 (1992); Trail et al., Science, 261:212(1993); and Rowlinson-Busza, et al., Curr. Opin. Oncol., 4:1142 (1992)).Tumor-directed delivery of these compounds enhances the therapeuticbenefit by, inter alia, minimizing potential nonspecific toxicities thatcan result from radiation treatment or chemotherapy. In another aspect,the compound of formula I and radioisotopes or chemotherapeutic agentscan be conjugated to the same anti-tumor antibody.

The characteristics of the agent itself and the formulation of the agentcan influence the physical state, stability, rate of in vivo release,and rate of in vivo clearance of the administered agent. Suchpharmacokinetic and pharmacodynamic information can be collected throughpreclinical in vitro and in vivo studies, later confirmed in humansduring the course of clinical trials. Thus, for any compound used in themethod of the invention, a therapeutically effective dose can beestimated initially from biochemical and/or cell-based assays.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe “therapeutic index,” which typically is expressed as the ratioLD50/ED50. Compounds that exhibit large therapeutic indices, i.e., thetoxic dose is substantially higher than the effective dose, arepreferred. The data obtained from such cell culture assays andadditional animal studies can be used in formulating a range of dosagefor human use. The dosage of such compounds lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity.

For the methods of the invention, any effective administration regimenregulating the timing and sequence of doses can be used. Doses of theagent preferably include pharmaceutical dosage units comprising aneffective amount of the agent. As used herein, “effective amount” refersto an amount sufficient to modulate PI3Kbeta expression or activityand/or derive a measurable change in a physiological parameter of thesubject through administration of one or more of the pharmaceuticaldosage units. “Effective amount” can also refer to the amount requiredto ameliorate a disease or disorder in a subject.

Suitable dosage ranges for the compounds of formula I vary according tothese considerations, but in general, the compounds are administered inthe range of 10.0 μg/kg-15 mg/kg of body weight; 1.0 μg/kg-10 mg/kg ofbody weight, or 0.5 mg/kg-5 mg/kg of body weight. For a typical 70-kghuman subject, thus, the dosage range is from 700 μg-1050 mg; 70 μg-700mg; or 35 mg-350 mg per dose, and two or more doses may be administeredper day. Dosages may be higher when the compounds are administeredorally or transdermally as compared to, for example, i.v.administration. In certain embodiments, the treatment of cancerscomprises oral administration of up to 750 mg/day of Compound I. Thereduced toxicity of this compound permits the therapeutic administrationof relatively high doses. For treatment of many solid tumors, a dosageof about 50-100 mg per dose, administered orally once or preferably atleast twice per day, is often suitable. In some embodiments, compound Iis administered orally, in three to five doses per day, using 20-150 mgper dose for a total daily dose between about 60 and 750 mg. In someembodiments, the total daily dose is between 100 and 500 mg, and in someembodiments the normalized daily dosage (adjusted for subject's bodyweight) is up to about 60 mg per kg of the treated subject's bodyweight.

The compounds may be administered as a single bolus dose, a dose overtime, as in i.v. or transdermal administration, or in multiple dosages.For IV or transdermal delivery, a dosage may be delivered over aprolonged period of time, and may be selected or adjusted to produce adesired plasma level of the active compound. In some embodiments, thedesired level will be at least about 1 micromolar, or at least about 10micromolar.

When the compound is administered orally, it is preferably administeredin two or more doses per day. In some embodiments, three doses per dayare administered. In some embodiments four doses per day areadministered.

Dosing may be continued for one day or for multiple days, such as about7 days. In some embodiments, daily dosing is continued for about 14 daysor about 28 days. In some embodiments, dosing is continued for about 28days and is then discontinued for about 7 days; the efficacy of thetreatment can be assessed during the break, when treatment with compoundI has been stopped, and if the assessment shows that the treatment isachieving a desired effect, another 7-28 day cycle of treatment withCompound I can be initiated.

Depending on the route of administration, a suitable dose can becalculated according to body weight, body surface area, or organ size.The final dosage regimen will be determined by the attending physicianin view of good medical practice, considering various factors thatmodify the action of drugs, e.g., the agent's specific activity, theidentity and severity of the disease state, the responsiveness of thepatient, the age, condition, body weight, sex, and diet of the patient,and the severity of any infection. Additional factors that can be takeninto account include time and frequency of administration, drugcombinations, reaction sensitivities, and tolerance/response to therapy.Further refinement of the dosage appropriate for treatment involving anyof the formulations mentioned herein is done routinely by the skilledpractitioner without undue experimentation, especially in light of thedosage information and assays disclosed, as well as the pharmacokineticdata observed in human clinical trials. Appropriate dosages can beascertained through use of established assays for determiningconcentration of the agent in a body fluid or other sample together withdose response data.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agent and the route of administration. Dosage and administration areadjusted to provide sufficient levels of the active moiety or tomaintain the desired effect. Accordingly, the pharmaceuticalcompositions can be administered in a single dose, multiple discretedoses, continuous infusion, sustained release depots, or combinationsthereof, as required to maintain desired minimum level of the agent.Short-acting pharmaceutical compositions (i.e., short half-life) can beadministered once a day or more than once a day (e.g., two, three, orfour times a day). Long acting pharmaceutical compositions might beadministered every 3 to 4 days, every week, or once every two weeks.Pumps, such as subcutaneous, intraperitoneal, or subdural pumps, can bepreferred for continuous infusion.

Subjects that will respond favorably to the method of the inventioninclude medical and veterinary subjects generally, including humanpatients. Among other subjects for whom the methods of the invention isuseful are cats, dogs, large animals, avians such as chickens, and thelike. In general, any subject who would benefit from a compound offormula I is appropriate for administration of the invention method.

The biological data disclosed herein was produced using a sample ofCompound I that contains less than 1% of the R isomer and >99% ^(S)enantiomer, as determined by chiral HPLC using a 4.6×250 mm ChiralcelOD-H column operated at 40° C., using a flow rate of 1 mL/min of 90:10hexanes/ethanol. This material was prepared as summarized in FIG. 9. Thematerial was characterized by HPLC to be over 99% pure (according toboth 214 nm and 254 nm UV detection), and was also characterized by nmrand electrospray mass spectroscopy. It was a white powder.

The material used in the Examples had the following characteristics:

Test Test Result Appearance White powder ¹H-NMR Consistent withstructure HPLC Assay 99+ % Chiral Purity (HPLC) 99.2% ee (99.6:0.4 ratioof S:R isomers)

Example 1 Chick Embryo Fibroblast Transformation Assay

Chick embryo fibroblasts (CEF) are transduced with viral stocks withversions of the human genes for the individual PI3K isoforms p110α,p110β, p110δ and p110γ. These transduced CEF lines are then plated in agrowth medium where oncogenically transformed cells form foci that canthen be stained and counted. Compound I inhibited the formation oftransformed foci in CEF cells that had been transduced with p110β withan EC50 of 150 nM. In contrast Compound I did not inhibit CEF cellstransduced with p110α significantly at the highest concentration tested(2000 nM). Denley A, Kang S, Karst U and Vogt PK, “Oncogenic signalingof class 1 PI3K isoforms.” Oncogene (2008) 27: 2561-2574. FIG. 3illustrates the readout of this assay.

Example 2 Preparation of Compound I

Compound I was synthesized by the route depicted in FIG. 5, usingmethods known in the art including adaptations of methods described inZhichkin, et al., Organic Letters, vol. 9(7), 1415-18 (2007), and U.S.Pat. No. 6,800,620.

Example 3 Effect of Compound I on Ovarian Cancer Cell Xenografts

Female nu/nu mice bearing OVCAR-3 xenografts (human ovarian cancercells) were maintained until tumor volume measured about 100 mm³. Atthat point, treatment began with compound I at a rate of 30 mg/kgadministered twice per day. Results of tumor volume measurements over a36 day period are shown in FIG. 8, and demonstrate that not only wastumor growth inhibited, but the size of the existing tumor was actuallyreduced by treatment with Compound I.

Example 4 Effect of Compound I on Renal Cancer Xenografts

Female nu/nu mice bearing A498 xenografts (human renal cancer cells)were maintained until tumor volume measured about 100 mm³. At thatpoint, treatment began with compound I at a rate of 30 mg/kgadministered twice per day. Results of tumor volume measurements over a20 day period are shown in FIG. 9, which demonstrates that this dosinglevel provides a significant reduction in tumor growth in vivo.

Example 5 Plasma Levels of Compound I in Mice Carrying Tumor Xenografts

Plasma levels of Compound I were observed in female nu/nu mice carryingone of the cancer cell xenografts used in the preceding two examples.Compound I was administered in a single dose at a rate of 30 mg/kg toeach test subject, and plasma levels were monitored for 12 hoursthereafter. Plasma levels of Compound I peaked around 2-4 hours afteradministration in each case, and had essentially returned to zero 8hours after the single dose at this rate, as shown in FIG. 10. The peakplasma concentration for these subjects, after a single injection at thedose shown to be effective for inhibiting tumor growth of each xenograft(see the preceding examples, and FIGS. 8-9) were generally below about7000 ng/mL.

Example 6 Pharmacokinetics and Toxicokinetics of Compound I in Rats

Compound I was dosed at 60, 120, or 240 mg/kg/day administered as asingle dose for up to 14 days in healthy rats. FIG. 11 shows themeasured blood levels of Compound I over a 24-hour period for each testsubject during the first day of treatment (dashed lines) and the lastday of treatment (solid lines) for the tolerated doses. The peakconcentration of compound I (Cmax) and area under the curve (AUC) fortolerated doses were higher than those observed with effective doses ofCompound I in the xenograft model tumors in mice. For example, the 60mg/kg per day dosing produced a Cmaxof 7300 ng/mL, while the Cmaxforeffective antitumor doses in the xenograft test were 2800 and 5600ng/mL. Similarly, the AUC for the 60 mg/kg/day dosing in this study was58,000 ng-h/mL, while the corresponding AUC in the xenograft bearingmice receiving effective treatment doses were 15,000 and 18,000 ng-h/mL.

1. A method of treating a solid tumor in a subject comprisingadministering to said subject an optically active compound of formula I

a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising an optically active compound of Formula I or apharmaceutically acceptable salt thereof; wherein the amount of thecompound of Formula I or its salt is an amount effective to treat thesolid tumor.
 2. The method of claim 1, wherein the solid tumor isselected from the group consisting of pancreatic cancer; bladder cancer;colorectal cancer; breast cancer; prostate cancer; renal cancer;hepatocellular cancer; lung cancer; ovarian cancer; cervical cancer;gastric cancer; esophageal cancer; head and neck cancer; melanoma;neuroendocrine cancers; CNS cancers; brain tumors; bone cancer; and softtissue sarcoma.
 3. The method of claim 1, wherein the solid tumor isselected from non-small cell lung cancer, small-cell lung cancer, coloncancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostatecancer and breast cancer.
 4. The method of claim 1, wherein theS-enantiomer predominates over the R enantiomer by a ratio of at leastabout 9:1.
 5. The method of claim 1, wherein the S-enantiomerpredominates over the R enantiomer by a ratio of at least about 19:1. 6.The method of claim 1, wherein the compound is administered orally. 7.The method of claim 1, wherein the compound is administered in solidform.
 8. The method of claim 7, wherein the solid form comprises theoptically active compound of Formula I admixed with at least onepharmaceutically acceptable excipient.
 9. The method of claim 7, whereinthe solid tumor is ovarian, renal, breast, lung, colon or prostatecancer.
 10. The method of claim 1 wherein the subject is refractory tochemotherapy treatment, or in relapse after treatment with chemotherapy.11. The method of claim 1 wherein the compound of formula I isadministered at a dose of 20-500 mg/day.
 12. The method of claim 1wherein the compound of formula I is administered at a dose of 50-250mg/day.
 13. The method of claim 1 wherein the compound of formula I isadministered at a dose of 50-150 mg twice per day.
 14. The method ofclaim 1 wherein a compound of formula I is administered at least twicedaily.
 15. The method of claim 1, further comprising reducing the levelof PI3Kδ activity in said subject.
 16. The method of claim 1, whereinthe subject is a human subject.
 17. The method of claim 16, wherein theconcentration of the compound in the blood is between 40-3000 ng/mL overa 12 hour period from the time of administration.
 18. The method ofclaim 16, wherein the concentration of the compound in the blood isbetween about 100 nM and 2000 nM in the treated subject.
 19. The methodof claim 1, wherein the agent is administered orally, intravenously orby inhalation.
 20. The method of claim 1, further comprisingadministering in addition to a compound of formula I to said subject, atherapeutically effective amount of at least one therapeutic agentand/or therapeutic procedure selected to treat said solid tumor in saidsubject.
 21. The method of claim 20, wherein said therapeutic agent isselected from the following group consisting of Docetaxel, Mitoxantrone,Prednisone, Estramustine, Anthracyclines, (doxorubicin (Adriamycin),epirubicin (Ellence), and liposomal doxorubicin (Doxil)), Taxanes(docetaxel (Taxotere), paclitaxel (Taxol), and protein-bound paclitaxel(Abraxane)), Cyclophosphamide (Cytoxan), Capecitabine (Xeloda) and 5fluorouracil (5 FU), Gemcitabine (Gemzar), methotrexate, Vinorelbine(Navelbine), an EGFR inhibitor such as erlotinib, Trastuzumab,Herceptin, Avastin, Platins (cisplatin, carboplatin), Temazolamide,Interferon alpha, and IL-2.
 22. The method of claim 20, wherein in saidtherapeutic agent is selected from the group consisting of an EGFRinhibitor, an mTOR inhibitor, a platin, and a taxane.
 23. The method ofclaim 20, wherein said therapeutic procedure is selected from the groupconsisting of peripheral blood stem cell transplantation, autologoushematopoietic stem cell transplantation, autologous bone marrowtransplantation, antibody therapy, biological therapy, enzyme inhibitortherapy, total body irradiation, infusion of stem cells, bone marrowablation with stem cell support, in vitro-treated peripheral blood stemcell transplantation, umbilical cord blood transplantation, immunoenzymetechnique, immunohistochemistry staining method, pharmacological study,low-LET cobalt-60 gamma ray therapy, bleomycin, conventional surgery,radiation therapy, high-dose chemotherapy and nonmyeloablativeallogeneic hematopoietic stem cell transplantation.
 24. The method ofclaim 1 further comprising obtaining a biological sample from saidsubject; and analyzing said biological sample with an analyticalprocedure selected from the group consisting of blood chemistryanalysis, chromosomal translocation analysis, needle biopsy,fluorescence in situ hybridization, laboratory biomarker analysis,immunohistochemistry staining method, flow cytometry or a combinationthereof.
 25. The method of claim 24, wherein the compound isadministered twice daily for about 28 days, and is then discontinued forat least 7 days.